title: Identification, characterization, and ontogenic study of a catechol O-methyltransferase from zebrafish.
abstract: To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5'- and 3'-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template, the cDNA encoding the zebrafish COMT was PCR-amplified. The recombinant zebrafish COMT protein was subsequently expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. Upon enzymatic characterization, purified COMT displayed methylating activity toward dopamine, dopa, and catecholestrogens, as well as three representative catechol drugs, methyldopa, dobutamine, and isoproterenol. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.
authors.affiliation: Department of Pharmacology, College of Pharmacy, The University of Toledo, Toledo, OH 43614, USA.
authors.forename: Adnan
authors.initials: A
authors.lastname: Alazizi
authors.forename: Ming-Yih
authors.initials: MY
authors.lastname: Liu
authors.forename: Frederick E
authors.initials: FE
authors.lastname: Williams
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Kurogi
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Sakakibara
authors.forename: Masahito
authors.initials: M
authors.lastname: Suiko
authors.forename: Ming-Cheh
authors.initials: MC
authors.lastname: Liu
mesh_terms: D000818:Animals; D001483:Base Sequence; D002394:Catechol O-Methyltransferase / Q000235:genetics* / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D057075:Enzyme Assays; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D050217:Metabolic Detoxication, Phase II / Q000235:genetics / Q000502:physiology*; D008745:Methylation; D023421:Models, Animal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015262:Xenobiotics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002396:Catechols; D015262:Xenobiotics; D029961:Zebrafish Proteins; D002394:Catechol O-Methyltransferase